Lipase is a carboxylic ester hydrolase and is widely used for food and surfactant industry as well as the synthesis of various chiral compounds. Among many microorganism-originated lipases, Candida antarctica—originated lipase B (referred as ‘CALB’ hereinafter) is composed of 317 amino acids and gets into the form of α/β hydrolase.
CALB is industrially very important in the selection of optical isomers and in the synthesis of polyester (Anderson et al., Biocatal. Biotransform., 1998, 16, 181), and can be used in variety of production of medical supplies and agricultural chemicals owing to the ability of optical conversion specifically with sec-alcohol and sec-amine (Rotticci et al., Chembiochem., 2001, 2, 766).
Recently, studies to express foreign proteins on the cell surface of unicellular organism such as yeast, bacteria including bacteriophage, etc. have been actively undergoing and applied for the production of a new vaccine, the screening of antigens and antibodies, the fixation of useful enzymes onto cell surfaces, and so on. For example, researches on the expression of proteins on cell surface have been progressed using a kind of yeast (Saccharomyces cerevisiae). This yeast induces the secretion of a useful foreign protein in a medium without losing its activity by taking advantage of intracellular secretion system of higher eukaryotic cells, which makes the yeast a useful candidate as a host cell for the production of important foreign proteins by genetic recombination techniques. α-agglutinin, a widely known cell wall protein, has been a major target for the surface expression of yeast (Schreuder et al., Yeast., 1993, 9, 399). Owing to α-agglutinin or other cell wall proteins, various enzymes such as α-galactosidase, glucoamylase, lipase, cutinase, etc. could be stably expressed on the cell surface.
In addition, according to a recent report, the development of industrially effective biocatalyst comes true by the simultaneous expression of various enzymes in a cell (Murai et al., Appl. Microbiol. Biotechnol., 1999, 51, 65). As of today, microorganism-originated biocatalysts for the biological conversion for the production of food or medical supplies have been made by the steps of crushing cells, separating the expressed enzyme, and fixing the enzyme onto carrier or treating with penetrating solvent such as toluene. But, such process has a problem of low productivity because of high costs and inactivity of the enzyme.
Recently, studies on the screening method using Saccharomyces cerevisiae, which is adequate for the expression of target proteins on the cell surface have been undergoing. As an eukaryotic cell, yeast has a similar protein production process to higher animals, and has enough size for the cell selection by FACS (fluorescence activated cell sorter), which can differentiate cells with delicate differences (VanAntwerp and Wittrup., Biotechnol. Prog., 2000, 16, 31). Using yeast expression system, manipulation of genes and a library construction can be simplified. Also, the difference of expression level can be overcome. It is also possible to select an enzyme which is most suitable for the surface expression, by screening with surface display system. The enzyme selected by this method can greatly enhance the use of the surface-expressed enzyme. While screening of yeast strains displaying antibodies, antigens or T-cell receptors with increased affinity using FACS has been reported a lot (Schreuder et al., Vaccine., 1996, 14, 383, Kieke et al., Protein. Eng., 1997, 10, 1303, Kieke et al., Proc. Natl. Acad. Sci. U S A., 1999, 96, 5651), the screening of strains displaying enzymes with increased activity has not yet been reported. Only the case that screening of carboxymethyl cellulase with increased activity by surface display system using ice nucleation protein originated from Pseudomonas sp has been reported so far (Kim et al., Appl. Environ. Microbiol., 2000, 66, 788).
The present inventors isolated novel cell wall attachment-mediating proteins from the industrial yeast, Hansenula polymorpha and developed a novel surface display system expressing a target protein on a cell surface using the same (PCT/KR00/00819). The Hansenula polymorpha is an industrially effective strain which has strong inducible promoters, has a stability both in high temperature and organic solvents, grows fast and produces foreign recombinant proteins very well. So, the strain is very suitable for the biological reaction system and the production of enzyme like lipase.
In order to mass-produce lipase, a useful biological catalyst, the present inventors have constructed Candida antarctica lipase B mutant library using a surface expression system (PCT/KR00/00819) expressing a target protein on a cell surface, and have selected a mutant strain having an excellent lipase activity from the library, and finally have completed this invention by developing a novel method for mass-production of mutant lipase protein from the yeast strain.